1. Field of the Invention
Streptococcus iniae is a causative agent for streptococcal disease which in turn affects a variety of cultured and wild fish and results in severe economic loss. In the United States, tilapia and hybrid striped bass production is estimated to lose 10 million dollars annually as the result of disease caused by this organism. Antibiotic treatment is ineffective and the need for a vaccine to control streptococcal disease is paramount.
This invention relates to the creation of a novel vaccine against Streptococcucs iniae which, due to its specific antigenic composition, does in fact provide superior protection over existing commercial vaccines.
2. Description of the Prior Art
Tilapia (Oreochromis niloticus) production is subjected to heavy economic losses due to mortality caused by Streptococcucs iniae. Antibiotic treatment is ineffective and the need for a vaccine is now paramount for the control of streptococcal disease. Vaccines have previously been developed against various Streptococcus and Enterococcus species utilizing strategies based on either intraperitoneal or intramuscular injection. Akhlaghi et al. (Comparison of Passive and Active Immunization of Fish against Streptococcus (Enterococcus);Journal of Fish Diseases;19:251-258;1996) demonstrated that passive immunization of fish was possible using antibodies against Streptococcus generated in sheep, rabbits and fish, however, this protection was of short duration (one month). Their studies suggest the importance of antibody in protective immunity to streptococcal disease. Few studies have examined antibody response prior to challenge (i.e. post vaccination). Eldar et al. (Development and Efficacy of a Vaccine against Streptococcucs iniae Infection in Farmed Rainbow Trout; Vet. Immunol. Immunopathol. 1997 56:175-183) did measure antibody titer by agglutination in rainbow trout vaccinated with 3.0xc3x971011 CFU S. iniae/ml. Titers detected were low (1:20-30) and were only 1:1 after six months. However, they suggested protection was due to this weak but measurable humoral response. This formalin-killed S. iniae vaccine protected trout in both experimental and field conditions for up to four months.
A S. difficile formalin-killed vaccine was reported to protect tilapia against challenge with S. difficile (Eldar et al.; Vaccination with Whole-cell Vaccine and Bacterial Protein Extract Protects Tilapia Against Streptococcus difficile Meningocephalitis; Vaccine. 13(9) 867-870; 1995 and Bercovier et al., Immunization with Bacterial Antigens: Infections with Streptococci and Related Organisms; Fish Vaccinology, Dev. Biol. Stand. Vol. 90 (Liiehaug, G., Midlyng, P J and Brown, F. eds.) Karger, Basel, Switzerland pp. 153-160, 1997).
We have now discovered a means for the creation of novel vaccines that are safe and effective for the control of Streptococcucs iniae in tilapia (Oreochromis niloticus). The vaccines comprise one or more killed isolates of Streptococcucs iniae in the form of whole cells and concentrated extracellular products having molecular weights greater than 2 kDa. These vaccines are effective in providing long lasting acquired immunity in tilapia to Streptococcucs iniae. 
In accordance with this discovery, it is an object of the invention to provide a novel, highly protective, vaccine against Streptococcucs iniae in tilapia.
It is also an object of this invention to provide both monovalent and polyvalent vaccines against Streptococcus iniae that are more efficacious than those previously used.
It is a further object of this invention to improve the viability and productivity of tilapia, striped bass and other fish species, and to reduce economic losses thereto caused by Streptococcucs iniae. 
Other objects and advantages of the invention will become readily apparent from the ensuing description.
Streptococcucs iniae isolates ARS-98-60 and ARS-98-T23 were deposited on Jan. 10, 2000 under the provisions of the Budapest Treaty in the Agricultural Research Service Culture Collection located at 1815 North University Street, Peoria, Ill. 61604, and have been assigned Deposit No.""s NRRL B-30238, and NRRL B-30242 respectively.
Streptococcucs iniae isolate ARS-98-10 was deposited on Feb. 25, 2000 under the provisions of the Budapest Treaty in the Agricultural Research Service Culture Collection located at 1815 North University Street, Peoria, Ill. 61604, and has been assigned Deposit No. NRRL B-30264.
xe2x80x9cVaccinexe2x80x9d is defined herein in its broad sense to refer to any type of biological agent in an administratable form capable of stimulating a protective immune response in an animal inoculated with the vaccine. For purposes of this invention, the vaccine may comprise one or more killed isolates of Streptococcucs iniae in the form of whole cells in combination with their concentrated extracellular products having molecular weights greater than 2 kDa. These vaccines are effective in controlling infection by Streptococcucs iniae in a variety of fish when administered thereto. Vaccination also significantly reduced abnormal behavior and morphology. Without being limited thereto, the vaccine is especially beneficial for the treatment of fish, both domestic and exotic, including yellowtail, rainbow trout, eels, striped bass and their hybrids, sea bass, sea bream, turbot and tilapia.
The starting material for use in preparing the vaccines of the invention may be any isolate of Streptococcucs iniae. Preferred strains include NRRL B-30238, and NRRL B-30242. While efficacy has been shown with both monovalent and polyvalent vaccines, polyvalent systems are preferred due to the indicated antigenic heterogeneity seen to exist in S. iniae. 
The inventive vaccine is a killed cell preparation or bacterin which includes a concentrated fraction of the extracellular filtrate (cell-free culture fluid) having a molecular weight greater than 2 kDa. While not wishing to be bound to theory, it is Applicants"" surmise that various low molecular weight components of the extracellular products of formalin killed S. iniae have an inhibitory effect upon the antigenicity of the bacterin suspensions. Concentration of the extracellular retentate thus substantially removes these inhibitory components and thus increases efficacy of the vaccine. To produce the vaccine, viable cells of S. iniae are exposed to formalin for a sufficient period of time to kill 100% of the cells. Typically formalin concentration would range from about 1% to about 5% (v/v), preferably from about 1% to about 3% (v/v).
Suitable exposure times for a particular formalin concentration to achieve 100% killing may be readily determined from lethal killing curves of % killed vs. time of treatment.
Propagation of S. iniae in preparation for treatment with formalin may be accomplished using conventional techniques and culture media known in the art.
Following culture in media, the cells are concentrated, for example, by centrifugation and the cell pellet and culture fluid separated. The filtrate, in the form of the cell-free culture fluid, is then concentrated by use of a 2 kDa molecular filter. Separation may be carried out to completion, with water added to resuspend the retentate. In the alternative, and presently preferred, separation may be carried out until there has been a 10 to 30 fold reduction in retentate volume, most preferably a 20 fold reduction in retentate volume. This leaves adequate water in the retentate so that suspension of the cell pellet occurs upon recombination with the retentate.
Appropriate ratios may be determinable by those skilled in the art, but are seen to typically range from about 5:1 (vol/vol) to about 15:1 (vol/vol), preferably about 10:1 (vol/vol).
Following resuspension of the cell pellet in the retentate, the killed S. iniae cells are prepared for administration by formulation in an effective immunization dosage to the fish. The dose may either be given as simply the retentate containing the resuspended killed cells, or may further include pharmaceutically acceptable carriers and adjuvants known in the art. An effective immunization dosage is defined herein as being that amount which will induce complete or partial immunity (elicit a protective immune response) in a treated animal against subsequent challenge with virulent S. iniae. Immunity is considered as having been induced in a population of treated animals when the level of protection for the population is significantly higher than that of an unvaccinated control group. The appropriate effective dosage can be readily determined by the practitioner skilled in the art. Typically, the vaccine will contain at least about 1xc3x97107 cells of S. iniae, preferably about 1xc3x97108 cells of S. iniae. Although greater amounts of cells may be administered, use of such higher levels is generally considered impractical.
The killed cells are prepared for administration by formulation in a pharmaceutically acceptable carrier such as water, physiological saline, mineral oil, vegetable oils, aqueous sodium carboxymethyl cellulose, or aqueous polyvinylpyrrolidone. The vaccine formulations may also contain optional adjuvants, antibacterial agents or other pharmaceutically active agents as are conventional in the art. Without being limited thereto, suitable adjuvants include but are not limited to mineral oil, vegetable oils, alum, and Freund""s incomplete adjuvant. Still other preferred adjuvants include microparticles or beads of biocompatible matrix materials. The microparticles may be composed of any biocompatible matrix materials as are conventional in the art, including but not limited to, agar and polyacrylate. The practitioner skilled in the art will recognize that other carriers or adjuvants may be used as well. For example, other adjuvants which may be used are described by Webb and Winkelstein [in Basic and Clinical Immunology, Stites et al. (ed.), fifth edition, Lange Medical Publications, Los Altos, Calif., 1984, pages 282-285], the contents of which are incorporated by reference herein.
In accordance with a preferred embodiment, the killed cells may be incorporated into microparticles or microcapsules to prolong the exposure of the antigenic material to the subject animal and hence increase the duration of protective immunity. The microparticles and capsules may be formed from a variety of well-known inert, biocompatible matrix materials using techniques conventional in the art. Without being limited thereto, suitable matrix materials include natural or synthetic polymers such as alginates, poly(lactic acid), poly(lactic/glycolic acid), poly(caprolactone), polycarbonates, polyamides, polyanhydrides, polyortho esters, polyacetals, polycyanoacrylates, polyurethanes, ethytlene-vinyl acetate copolymers, polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonated polyolefins, polyethylene oxide, and particularly agar and polyacrylates. Examples of techniques for incorporation of materials into microparticles or encapsulation which may be used herein are described by Sparks [Microencapsulation, In: Kirk-Othmer Encyclopedia of Chemical Technology, third edition, John Wiley and Sons, New York, (1981) volume 15, pages 470-493], Kydonius [Controlled Release Technologies: Methods, Theories, and Applications, CRC Press, Cleveland, Ohio, 1980], Gombotz et al. [U.S. Pat. No. 5,019,400], or Beck [U.S. Pat. No. 4,919,929], the contents of each of which are incorporated by reference herein.
The vaccines of the invention may be administered to the subject animal by any convenient route which enables the cells to elicit an immune response, such as by intraperitoneal or intramuscular injection. However, intraperitoneal injection is preferred for practical considerations. The vaccine may be administered in a single dose or in a plurality of doses. Dependent upon rearing conditions, the vaccine may be administered in multiple doses the timing of which would be readily determinable by the skilled artisan.